PROJECT SUMMARY Blood flow from the heart to the brain is strictly regulated to protect the delicate brain tissue, because improper blood flow can give rise to numerous cardiovascular diseases and brain injuries. TRPM4 is one of the major actors regulating blood flow in the vascular smooth muscle cells in the cerebral arteries when intracellular pressure changes. Mutation or dysfunction of TRPM4 is linked to numerous cardiovascular diseases, including stroke and Brugada syndrome. TRPM4 and its closest homolog, TRPM5, are Ca2+-activated, nonselective, voltage-gated ion channels. TRPM5 is highly expressed in pancreatic beta cells, and dysfunction or mutation in TRPM5 is associated in type II diabetes and obesity. In addition, TRPM4 and TRPM5 in the taste bud cells play an important role in taste signaling, and loss of both channels abolishes the ability to detect bitter, sweet, or umami stimuli. Taken together, TRPM4 and TRPM5 have a wide range of roles in physiology and pathophysiology. Both TRPM4 and TRPM5 belong to the TRPM (melastatin-like transient receptor potential) subfamily of the TRP superfamily, and they are the only two members impermeable to Ca2+. The lack of a canonical positively charged voltage-sensing domain makes a mystery of how TRPM4 and TRPM5 sense voltage. Despite sharing 45% amino acid identity, TRPM4 and M5 have distinct functional and pharmacological properties in terms of kinetics and sensitivities to drugs. A collaboration has been built between Takeda California, Inc. and our lab to study the important role of TRPM5 in treatment of diabetes. The high-affinity drugs specifically targeting TRPM5 provided by Takeda and the potential future drug development strengthen our proposal on studying the pharmacology of these two channels. At present, we do not understand, in molecular detail, how the channels are activated in a voltage-dependent manner, how they are modulated by small molecules binding to them at specific sites, how they are distinguished by various drugs, or how their channel functions are modulated by other proteins such as calmodulin. Building on the success of solving the first human TRPM4 structure in closed state (published in Nature), we propose to continue the cryo-EM studies of TRPM4 and TRPM5 and their pharmacology, combined with complementary electrophysiology experiments and collaboration with Takeda. The outcome of this proposal will define the molecular basis for the voltage-dependent gating activity of these ion channels, for ligand recognition, and for the action of modulators. These advances, in turn, will provide a foundation for developing new therapeutic agents against cardiovascular diseases and diabetes and for a deeper understanding of the function of the voltage-gated TRPM family members.